In utero hematopoietic stem cell transplantation: a caprine model for prenatal therapy in inherited metabolic diseases.

OBJECTIVES: We explored the feasibility and efficacy of in utero hematopoietic stem cell transplantation in the caprine animal model system with the objectives of determining procedures for transplantation and establishing methods for detecting engraftment. METHODS: Male fetal liver hematopoietic stem cells were injected into female fetuses during the immunotolerant period, using either hysterotomy or ultrasound-guided injections. RESULTS: The rate of fetal death was much lower for the ultrasound-guided injections. Donor cells were observed in the peritoneal fluid of 4 fetuses 3 days after injection, but no donor cells were detected in tissues at longer time periods. CONCLUSIONS: Ultrasound-guided injection of hematopoietic stem cells into the abdomen of a developing fetus is safe and feasible. The parameters required for successful engraftment have not yet been identified.

Biochemical and morphological expression of early prenatal caprine beta-mannosidosis.

Lysosomal storage diseases associated with early-onset pathological changes may require prenatal therapy to avert the profound effects of the metabolic error on organs, especially the central nervous system. The present investigation determined the extent of expression of beta-mannosidase deficiency in the caprine fetus at 62 days of gestation, near the end of the period of immunotolerance when donor cells can engraft in various organs without immune rejection and supply missing enzyme. Three pairs of obligate carrier goats from the beta-mannosidosis colony were mated. Out of six fetuses delivered at 62 days of gestation, one (V385) was identified by measurement of beta-mannosidase activity as the only fetus affected with beta-mannosidosis. Thin-layer chromatography and quantitation of oligosaccharides revealed the presence of tri- and disaccharides, typical of beta-mannosidosis, only in V385. Morphological analysis revealed cytoplasmic vacuolation typical of beta-mannosidosis in V385; in thyroid, spinal cord, and kidney, the pattern of vacuolation was similar to, but less severe than, that observed previously in newborn affected goats. On the basis of these results, it will be possible to determine the effects of prenatal cell transplantation therapeutic strategies performed during the period of immunotolerance by monitoring phenotypic characteristics after treatment.

mRNA levels for central nervous system myelin proteins in myelin deficiency of caprine beta-mannosidosis.

Caprine beta-mannosidosis is an inherited lysosomal storage disease that leads to a deficiency of oligodendrocytes and hypomyelination. Our previous results demonstrated that low levels of myelin-associated glycoprotein (MAG), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and proteolipid protein (PLP) found in CNS samples correlated with decreased yields of myelin. However, there was a relative preservation of myelin basic protein (MBP) in the spinal cord samples of affected goats. This report shows that the amounts of myelin protein mRNAs in the spinal cords of affected goats relative to control goats are also decreased. The levels of mRNA for MAG, MBP and PLP in affected goat spinal cords compared with those of controls were equally decreased to approximately 50% for the three myelin proteins. This suggests that the relative preservation of MBP protein in the spinal cords is not due to a higher MBP mRNA level, but might be due to a difference in post-transcriptional processes.

Development and efficacy of ultrasound-guided fetal fluid aspiration techniques for prenatal diagnosis of caprine beta-mannosidosis.

Ultrasound-guided fetal fluid sampling was performed on 13 pregnant goats at Days 59 to 65 of gestation to establish safe techniques for accurate sampling and to determine the feasibility of prenatal diagnosis of beta-mannosidosis. Fluids were analyzed for electrolyte and creatinine content to assess accuracy of sampling. Values correlated well with previously reported concentrations for caprine and ovine fetal fluids at the same gestational stage. The single abortion which occurred following ultrasound-guided sampling was correlated with placentome penetration and aspiration of bloody fluids. Thin layer chromatography of amniotic and allantoic fluids was performed to detect oligosaccharides that accumulate in beta-mannosidosis. Abnormal accumulated oligosaccharides were identified in the allantoic but not amniotic fluid from a beta-mannosidase-deficient 62-d-old fetus. Thus, allantocentesis was shown to be an optimal, safe procedure for providing information at this gestational stage to diagnose caprine beta-mannosidosis.

Biochemical and histochemical analysis of lysosomal enzyme activities in caprine beta-mannosidosis.

Goats affected with beta-mannosidosis, an autosomal recessive disease of glycoprotein catabolism, have deficient tissue and plasma levels of the lysosomal enzyme beta-mannosidase. Pathological characteristics include cytoplasmic vacuolation in the nervous system and viscera, and myelin deficits that demonstrate regional variation. This study was designed to determine the correlation between beta-mannosidase activity in normal animals and the severity of lesions in affected goats, and to assess the regional changes in lysosomal enzyme activity in specific regions and cell types in affected animals. Although enzyme activity in normal organs (kidney, thyroid, brain) is correlated in general with the accumulation of uncatabolized substrate and with the extent of vacuolation, this correlation does not extend to assessment of specific regions of the central nervous system (CNS). In affected goats, the activities of alpha-mannosidase, alpha-fucosidase, and beta-hexosaminidase are elevated to a greater extent in all CNS regions than in organs. The results suggest cell-specific, organ-specific, and enzyme-specific regulation of changes in lysosomal enzyme activity in the presence of metabolic perturbations, such as deficiency of beta-mannosidase activity.

Myelin-associated glycoprotein (MAG) in myelin deficiency of caprine beta-mannosidosis.

Caprine beta-mannosidosis is an inherited lysosomal storage disorder due to a deficiency of beta-mannosidase which cleaves beta-linked mannose residues from the ends of N-asparagine linked oligosaccharides of glycoproteins. Histological and chemical examination has revealed a deficiency of compact myelin in the brains and spinal cords of affected goats. Since myelin-associated glycoprotein (MAG) is glycosylated and its metabolism could be directly affected in this disease, we investigated the possibility of a differential treatment of MAG in caprine beta-mannosidosis in comparison to non-glycosylated myelin proteins. MAG, myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), proteolipid protein (PLP) and glial fibrillary acidic protein (GFAP) were quantified by western blot analysis in whole homogenates of spinal cords and hemispheres from affected goats at 1, 3 and 6 days of age and from normal controls. The yields of isolated myelin from the spinal cords of affected goats varied from 37 to 63% of normal and were 7% or less of normal from the hemispheres. In mutant spinal cords, the deficits of MAG, CNP and PLP measured in whole homogenates corresponded reasonably well with the decreased myelin yields, but the levels of MBP were consistently much closer to control levels than those of the other myelin proteins. A greater deficiency of PLP than MBP was also apparent in the myelin fractions purified from the affected spinal cords. In homogenates of mutant hemispheres, MAG, MBP, PLP and CNP were undetectable or at trace levels in comparison to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Caprine beta-mannosidosis: regional differences in deficits of CNS myelin proteins.

Caprine beta-mannosidosis is an autosomal recessive disorder characterized by marked deficiency of beta-mannosidase activity, accumulation of oligosaccharides, and pathologic changes involving prominent dysmyelination. The myelin deficits show marked regional variation, with spinal cord mildly affected and many brain regions severely affected by morphologic criteria. In this study, levels of myelin basic protein (MBP) and proteolipid protein (PLP) were measured by immunoblotting in samples prepared from spinal cord, brainstem and cerebral hemispheres of normal and affected goats at 2-4 days (newborns) and 2-4 weeks of age. In affected goats, total levels of MBP in spinal cord were normal, while PLP levels were 60-70% of normal at both ages. In contrast, PLP and MBP in brainstem and cerebral hemispheres were severely decreased at both ages, with levels of PLP 10-13% and MBP 25-29% of normal in newborns, and generally more reduced at 2-4 weeks. When myelin fractions were isolated on 0.32/0.85 M sucrose gradients, yields were about 38 and 25% of normal in spinal cord at the two ages, but less then 3% of normal in brainstem. Yields of myelin-like fraction were decreased as well, but to lesser extents than yields of myelin. Myelin from spinal cord had a normal composition with regard to PLP and MBP content, while the myelin fraction from brainstem was markedly deficient in both proteins. This suggests formation of myelin with a very abnormal composition in brainstem, or inclusion of large amounts of membranes other than myelin in this fraction. The more severe deficits in brainstem and cerebral hemispheres compared to spinal cord are consistent with morphologic observations.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropathology of bovine beta-mannosidosis.

beta-Mannosidosis, an inherited defect of glycoprotein catabolism previously identified in goats and humans, has been recently diagnosed in Salers cattle. This disorder is associated with deficiency of lysosomal beta-mannosidase and accumulation of oligosaccharides. Analysis of bovine beta-mannosidosis neuropathology was initiated to determine whether independently arising gene defects in cattle and goats result in expression of similar lesions. Brain, spinal cord, and selected peripheral nerves from seven affected newborn Salers calves and three normal newborn calves were available for gross, light microscopic, and electron microscopic analysis. Gross examination revealed hydrocephalus of variable severity and myelin deficiency in the cerebral hemispheres, cerebellum, and brainstem. Microscopic examination revealed cytoplasmic vacuolation, myelin deficiency, and axonal spheroids of similar type and distribution to that reported in affected goats. Cytoplasmic vacuolation resulting from lysosomal storage showed consistent variation among cell types. Myelin deficits were more severe in the cerebral hemispheres and cerebellum than in the spinal cord. Axonal spheroids occurred in the cerebrum, brainstem, cerebellum, and trigeminal nerve endings. The presence of similar lesions in bovine and caprine beta-mannosidosis supports a direct relationship with the gene defect.

Thyroid structure and function in bovine beta-mannosidosis.

In bovine beta-mannosidosis, the thyroid in the affected newborn shows marked cytoplasmic vacuolation. There is an associated reduction in the serum concentrations of thyroxine and tri-iodothyronine.

Evaluation of videodisc modules: a mixed method approach.

The purpose of this study was to evaluate the design and implementation of 10 neuropathology interactive videodisc instructional (IVI) modules used by Michigan State University medical students in the College of Osteopathic Medicine and the College of Human Medicine. The evaluation strategy incorporated a mixed method approach using qualitative and quantitative data to examine levels of student acceptance for the modules; ways in which IVI modules accommodate different learner styles; and to what extent the modules facilitate the attainment of higher level learning objectives. Students rated the units highly for learning effectiveness; many students reported group interaction as beneficial; and students expressed a desire for more IVI in the curriculum. The paper concludes with recommendations for future use of interactive videodisc technology in the teaching/learning process.

Regional central nervous system oligosaccharide storage in caprine beta-mannosidosis.

Goats affected with beta-mannosidosis, an autosomal recessive disease of glycoprotein metabolism, have deficient activity of the lysosomal enzyme beta-mannosidase along with tissue storage of oligosaccharides, including a trisaccharide [Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc] and a disaccharide [Man(beta 1-4)GlcNAc]. CNS myelin deficiency, with regional variation in severity, is a major pathological characteristic of affected goats. This study was designed to investigate regional CNS differences in oligosaccharide accumulation to assess the extent of correlation between oligosaccharide accumulation and severity of myelin deficits. The concentrations of accumulated disaccharide and trisaccharide and the activity of beta-mannosidase were determined in cerebral hemisphere gray and white matter and in spinal cord from three affected and two control neonatal goats. In affected goats, the content of trisaccharide and disaccharide in spinal cord (moderate myelin deficiency) was similar to or greater than that in cerebral hemispheres (severe myelin deficiency). Thus, greater oligosaccharide accumulation was not associated with more severe myelin deficiency. Regional beta-mannosidase activity levels in control goats were consistent with the affected goat oligosaccharide accumulation pattern. The similarity of trisaccharide and disaccharide content in cerebral hemisphere gray and white matter suggested that lysosomal storage vacuoles, more numerous in gray matter, may not be the only location of stored CNS oligosaccharides.

Caprine beta-mannosidosis. Abnormal thyroid structure and function in a lysosomal storage disease.

Deficient activity of the lysosomal enzyme beta-mannosidase leads to widespread tissue accumulation of oligosaccharides in caprine beta-mannosidosis, an autosomal recessive neurovisceral storage disease. Severe thyroid morphologic abnormalities found in a previous light microscopic survey of tissues from neonatal affected goats suggested the possibility of impairment of function. Since considerable evidence indicates that thyroid hormone plays an important role in regulation of myelination, thyroid hormone deficiency, if present during central nervous system development, could be a factor in the hypomyelination seen in affected animals. Thus, this study was designed to characterize thyroid structure and function in beta-mannosidosis. To investigate developmental aspects of structural abnormalities, thyroids from six pairs of affected and control animals ranging in age from 96/150 days gestation to 3 days postnatal were analyzed by light and electron microscopy. Major findings in thyroids from affected animals, as early as 96/150 days gestation, included follicle irregularities and pronounced presence of lysosomal storage vacuoles in all cell types, particularly in follicular cells. The degree of cytoplasmic vacuolation increased with advancing age. To assess thyroid function, thyroid hormone concentrations were determined in six age-matched, neonatal pairs of affected and control goats. Significantly decreased thyroid hormone concentrations were present in affected animals. It is hypothesized that thyroid hormone deficiency plays a role in the pathogenesis of hypomyelination in affected animals. This study comprises, to our knowledge, both the most complete description of developmental abnormalities and the first report of abnormal function in an endocrine organ in a lysosomal storage disease. Further, this report suggests that systemic perturbations induced by a genetically determined deficiency of a lysosomal hydrolase could be a factor in the pathogenesis of central nervous system lesions.

Investigation of dysmyelinogenesis in caprine beta-mannosidosis: in vitro characterization of oligodendrocytes.

Central nervous system myelin deficiency is a consistent feature of caprine beta-mannosidosis, an autosomal recessive neurovisceral lysosomal storage disease. To investigate the possibility of an intrinsic oligodendrocyte defect in beta-mannosidosis, oligodendrocyte-enriched glial cultures from the cerebral hemisphere white matter of two affected and six control goats were compared with respect to culture yield and morphology. Fewer oligodendrocytes were cultured per gram of white matter from affected animals than from control animals. Galactocerebroside-positive oligodendrocytes from all animals were similar morphologically at all stages of culture by phase contrast and fluorescence microscopy. These findings are consistent with in vivo morphological observations and suggest that differentiated oligodendrocytes from affected animals do not show morphological abnormalities in culture. However, increased numbers of galactocerebroside-negative bipolar cells, which may be glial progenitor cells, were present in cultures from affected animals. This observation suggests the possibility of a defect in differentiation to mature oligodendrocytes, with persistence of the undifferentiated glia during late stages of development.

Caprine beta-mannosidosis: development of glial and myelin abnormalities in optic nerve and corpus callosum.

In caprine beta-mannosidosis, an inherited dysmyelinating disorder, the myelin deficit shows substantial variation throughout the nervous system. In this study morphometric analysis of optic nerve and corpus callosum sections at selected developmental stages was conducted in order to investigate development and persistence of myelin sheaths, the population of axons ensheathed, and the extent of myelin deficits and glial cell abnormalities. The results show that the myelin deficit is severe at very early stages of development and persists to about the same extent into postnatal life. The corpus callosum, much more severely involved than the optic nerve, contains a substantially smaller percentage of myelinated axons when compared to control. In both regions, larger axons are preferentially myelinated. In the corpus callosum before myelination begins, many glial cells appear abnormal, suggesting an early cellular defect. In the postnatal, myelin-deficient corpus callosum, there is a substantial decrease in glial cell density as compared to control, with abnormal appearance of many of the remaining cell profiles. These results define developmental characteristics of the dysmyelination in caprine beta-mannosidosis and document both the early appearance and the persistence of glial cell body and myelin abnormalities.

Effect of nerve growth factor on the transplacental induction of neurinomas by ethylnitrosourea in Sprague-Dawley rats.

Administration of nerve growth factor (NGF) to the offspring of Sprague-Dawley rats transplacentally exposed to 50 mg/kg ethylnitrosourea on the 20th day of gestation resulted in a significant reduction of trigeminal and peripheral nerve neurinomas. Forty, 60, and 80 micrograms of NGF was administered in five s.c. doses, one dose on each of days 12-16, 90-94, and 210-214 postnatally. Of the 34 rats in the NGF-treated group, 11 animals were affected with trigeminal nerve neurinomas as compared to 18/34 in the NGF-untreated group (P less than 0.05). In the peripheral nerves (spinal cord nerve roots) there were five and 11 neurinomas, respectively, in each group of 34 rats. When the total numbers of neurinomas (trigeminal and peripheral nerves) between these groups were compared (16/34 versus 29/34), the significance of neurinoma reduction was P less than 0.01. Five trigeminal and two peripheral neurinomas in the NGF-untreated group were shown by immunohistochemical staining to contain nerve growth factor receptor protein, whereas none of the neurinomas in the NGF-treated group were positive for the receptor protein. The results obtained from this experiment lend support to the hypothesis that NGF has the capability to reduce the oncogenic consequences of ethylnitrosourea exposure perhaps by the process of maturation and/or differentiation of the transformed cells, and that this effect may depend upon the presence of receptor binding sites.

Otic pathology of caprine beta-mannosidosis.

Caprine beta-mannosidosis is an autosomal recessive defect of glycoprotein catabolism with a deficiency of tissue and plasma beta-mannosidase activity and tissue accumulation of oligosaccharides within lysosomes. This rapidly fatal genetic disorder of Nubian goats is expressed at birth by a variety of clinical signs including deafness. Affected goats had folded pinnas, and the tympanic cavity was decreased due to multiple, polypoid projections of bone covered by middle ear mucosa which obstructed the view of the cochlear promontory. Numerous cells of the cochlear duct including mesothelial and epithelial cells of Reissner's membrane, mesothelial cells lining the scala tympani, cells of the stria vascularis, numerous supportive cells of the organ of Corti, cochlear hair cells, endothelial cells, perithelial cells, fibroblasts, macrophages, and neurons of the spiral ganglion contained numerous nonstaining intracytoplasmic vacuoles which resulted in distention of affected cells and caused thickening of involved structures. Ultrastructurally, the vacuoles were membrane-bound and consistent with lysosomes. Vacuolated cells were desquamated into the scala vestibuli and scala tympani. This is one of few reports describing light and electron microscopic otic alterations of a storage disease. Goats with beta-mannosidosis appear to be good models of hearing loss in patients with storage disease.

Dysmyelinogenesis in caprine beta-mannosidosis: ultrastructural and morphometric studies in fetal optic nerve.

The optic nerves from a goat fetus affected with beta-mannosidosis and a control fetus were analysed morphologically in order to investigate developmental aspects of beta-mannosidosis-associated myelin deficits. In the affected fetus, the number of myelinated axons per unit area was about 25% of the control values. Histograms of axonal diameter indicated that a greater percentage of the myelinated and unmyelinated axons were of larger caliber in the affected fetus than in the control fetus and that very few small axons were myelinated in the affected animal. The mean values of myelin sheath thickness in the affected and control animals did not differ significantly. Ultrastructural analysis revealed a decreased proportion of oligodendrocytes and an increased proportion of astrocytes in the affected fetus. These results indicate that the pathogenetic process leading to cellular abnormalities and myelin deficits in beta-mannosidosis has been initiated prior to 124 days gestation, during an early stage of myelination in the goat optic nerve. The decrease in number of oligodendrocytes suggests that early cell death and/or change in oligodendrocyte proliferation contribute to the myelin deficit. Analysis of the prenatal development of lesions will help clarify the pathogenesis of dysmyelinogenesis in beta-mannosidosis.

Caprine beta-mannosidosis: phenotypic features.

The clinical features of caprine beta-mannosidosis were evaluated in 10 newborn goats, one stillborn goat and one goat fetus. The phenotypic abnormalities observed in all 10 live affected animals included an inability to rise from a recumbent position, moderate to marked intention tremor, eye movements resembling pendular nystagmus, clinical deafness, bilateral Horner's syndrome, carpal contractures, pastern joint hyperextension, thickened skin and to a varying degree, a dome-shaped skull. Subjective evaluation suggested that most animals had a decreased muscle mass. Together, these characteristics represent a common phenotype which is expressed at birth in caprine beta-mannosidosis.